INTRODUCTION

Mitogen-activated protein kinases (MAPK)¹ transduce signals from cell membrane to nucleus in response to a wide variety of stimuli (1-3). Four groups of MAPKs have been identified in mammalian cells: the extracellular signal-regulated kinases (ERK) (also referred to as p42/44 MAPK) (1, 4, 5), the c-Jun N-terminal kinases (JNK) or stress-activated protein kinases (SAPK) (6-12), p38/CSBP/RK/MPK2/MXI2 (13-16), and ERK5 kinase (17). The mammalian ERKs are activated by growth factors and mitogenic stimuli (1, 4), whereas p38 and JNK are regulated by stress-inducing signals (i.e. UV irradiation, osmotic shock) and by proinflammatory cytokines (i.e. interleukin-1 (IL-1) and tumor necrosis factor  (TNF) (6-14, 18)).

MAPKs are activated through phosphorylation on both threonine and tyrosine residues at the Thr-Xaa-Tyr dual phosphorylation motif (18-22). This motif is located in kinase subdomain VIII where Xaa is a Glu, Pro, and Gly for the ERK (19, 20, 22), JNK (6, 14), and p38 (13, 18) group of kinases, respectively. Activation of MAPK is mediated by dual specificity MAPK kinases, MKK or MEK (23-28). MEK1 and -2 catalyze the phosphorylation of ERK1/2 (23, 24), whereas MKK4/SEK1 mediates the activation of JNK and p38 (25-27). MKK3 and MKK6 specifically activate p38 (26-32). Once activated, MAPK phosphorylates several transcription factors at serine and threonine residues, thereby regulating gene expression. Each group of MAPK appears to have different substrate specificity. JNK phosphorylates transcription factors c-Jun (6), ATF-2 (33), and ELK-1 (34), whereas p38 phosphorylates ATF-2 (18), MEF2C (35), and CHOP-1 (36). In addition, p38 phosphorylates and activates MAPK-activated protein (MAPKAP) kinase-2 and -3 (15, 37). Upon activation by p38, MAPKAP kinase-2 phosphorylates the small heat shock proteins HSP25/27 (15).

p38 was originally identified in lipopolysaccharide (LPS)-stimulated mouse macrophages and was found to have substantial homology to the Saccharomyces cerevisiae HOG1 kinase (13, 38). The human homologues of p38 were cloned after p38 was identified with a radiophotoaffinity-labeled pyridinyl imidazole compound (14). Inhibition of p38 by this class of compound prevents the production of IL-1 and TNF by human monocytes stimulated with LPS (14). In addition to the original isoform of p38 (now referred to as p38), a second p38 kinase member (p38) was identified which shows 74% amino acid identity to p38 (39). p38 also has a TGY motif in kinase subdomain VIII (39). More recently, a third p38 kinase family member with a TGY motif was cloned and is termed p38/ERK6/SAPK3 (40-42). The amino acid sequence of p38/ERK6/SAPK3 is 60% identical to p38 (40).

Here we report the isolation of a novel p38 MAPK (p38) with a TGY motif in its activation domain. p38 was characterized with regard to tissue distribution, stimulus activation, MKK activation, substrate specificity, and inhibitor sensitivity. These studies reveal interesting similarities as well as differences in the properties of p38 as compared with p38.

EXPERIMENTAL PROCEDURES

Recombinant GST-c-Jun protein was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Recombinant PHAS-I protein was purchased from Stratagene (La Jolla, CA). ATF-2 was amplified by PCR from human skeletal muscle cDNA using two primers (5-CATATGCAATACAAGGACCTGTGGAAT-3 and 5-CCTCCGCTCGAGTTATAGAGGCATTTTTTTAATGTCATC-3) and cloned into bacterial expression vector pAMG21. Recombinant protein was expressed in Escherichia coli strain FM15 and purified by S-Sepharose and Q-Sepharose chromatography.

MAPKAP kinase-2 was amplified by PCR from human monocyte cDNA using two primers (5-ACAACAGGATCCCAGATCAAGAAGAAC GCCATC-3 and 5-ACAACACTCGAGTCCTGTAGAGAGTTATTGCTT-3). MAPKAP kinase-3 was amplified by PCR from a human lung cDNA library using two primers (5-CTCGCTGAATTCGATGGTGAAACAGCAGAGGAGCAG-3 and 5-CCGGAGGTCGACCTACTGGTTGTTGCAGCCCTGTG-3). The resulting PCR products were cloned separately into a GST fusion vector, pGEX-4T (Pharmacia Biotech Inc.). GST-MAPKAP kinase-2 and -3 were expressed in E. coli strain BL2/DE3 (Pharmacia), and fusion protein was purified over a glutathione column (Pharmacia). MKK3 was amplified by PCR from human skeletal muscle cDNA (CLONTECH, Palo Alto, CA) with two primers (5-ACAACAATCTAGAAGGAGGAATAACATATGGCTCATCATCATCATCATCATTCCAAGCCACCCGCACCCAC-3 and 5-TCCCGCTCGAGCTATGAGTCTTCTCCCAGGAT-3) and then cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). To generate constitutively active MKK3 (ca-MKK3), site-directed mutagenesis (43) was used to replace Ser-189 and Ser-193 with Glu. This DNA was cloned into the baculovirus transfer vector pVL1392 (Invitrogen) and expressed in Hi-5 cells. Recombinant ca-MKK3 was purified by hydroxyapatite (Bio-Rad) followed by Phenyl-Sepharose HP (Pharmacia) chromatography. To generate human FLAG-tagged p38, two primers from the published nucleotide sequences (14) were used in PCR with human peripheral blood leukocyte cDNA as templates. The PCR product was then cloned into mammalian expression vector pCMVXV5. HA-tagged MKK6 in pME vector (31) was kindly provided by Dr. Hagiwara Masatoshi and HA-tagged MKK3 and MKK4 (SEK1) in mammalian expression vector pMT (44) were provided by Dr. James Woodgett.

Molecular Cloning of p38

An expressed sequence tag (EST) (311 base pairs) with homology to p38 was identified in the Amgen EST data base. Gene-specific forward and reverse primers were designed from the EST sequence and used in PCR to clone full-length cDNA with the Marathon-Ready human fetal brain cDNA templates (CLONTECH) following the manufacturer's protocol. These Marathon-Ready cDNAs have adaptors ligated at the 5 and 3 ends. The gene-specific forward primer (5-GAGCTGTCCAAGACCTACGTGTC-3) and an adaptor primer (CLONTECH) were used in combination to amplify the 3 portion of p38. The gene-specific reverse primer (5-CTGGGGTGAAGACATCCAGG-3) and the adaptor primer were used to amplify the 5 portion of p38. PCR was performed for 30 cycles (95 °C for 30 s, 42 °C for 30 s, and 72 °C for 20 s) followed by an extension at 72 °C for 7 min. The resulting PCR product was ligated into the pCR2.1 vector (Invitrogen) and sequenced on both strands. A second murine EST sequence (GenBank^TM accession number W53837) that has homology to the Amgen EST sequence was identified in the GenBank data base. This EST fragment was used as a probe to screen a human macrophage library, and two clones were isolated. Sequencing of one of the clones revealed an identical open reading frame as the one cloned by PCR. The clone isolated from human fetal brain library was used for subsequent studies described here.

Full-length p38 cDNA was cloned into a mammalian expression vector PCR3.1 (Invitrogen) by PCR using two primers (5-ACCATGGACTACAAGGACGACGATGACAAGAGCCTCATCCGGAAAAAGGGCTTCTACAAG-3 and 5-ACCTGCAGGCGATTCTCCAGAT-3). The first primer added a FLAG epitope at the 5 end. PCR site-directed mutagenesis (43) was used to create a p38 mutant (AGF) by substituting Thr-180 and Tyr-182 with an Ala and a Phe, respectively. The inserts were completely sequenced to make sure that no PCR errors were introduced.

Northern and Dot-Blot Analysis of p38 mRNA

A Northern blot filter containing poly(A)⁺ RNA from multiple tissues and a normalized Master blot filter containing mRNA from 50 different tissues (CLONTECH) were probed with a ³²P-labeled DNA fragment generated from the 5 portion of the coding region of p38 (nucleotides 1 to 550). Hybridization was performed at 68 °C in ExpressHyb Buffer (CLONTECH) followed by two washes in 0.1 × SSC, 0.1% SDS at 55 °C. Blots were exposed overnight at 70 °C. The same Northern blot was then probed with a ³²P-labeled DNA fragment generated from the 5 portion of the coding region of p38 using identical hybridization and washing conditions.

293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin. For transfection, 2 × 10⁶ cells were plated onto 100-mm dishes 16-20 h before transfection. DNA (2.0 µg of p38, 8.0 µg of all other DNAs) was transfected into 293 cells using LipofectAMINE^TM (Life Technologies, Inc.). Transfected cells were incubated for 5 h in serum-free DMEM, further incubated in DMEM with 10% fetal calf serum, and harvested 48 h after transfection.

Immunoprecipitation was performed as described previously (45). Briefly, cells were dislodged into lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% Igepal, 150 mM NaCl, 20 mM NaF, 0.2 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA) and sedimented (15,000 × g for 60 min) to remove insoluble debris. Total protein in cell lysates was quantified by the Bradford method using a protein assay kit (Pierce). Supernatants containing 100 µg of protein were immunoprecipitated with 5 µg of anti-HA mAb 12CA5 (Berkeley Antibody Co., Berkeley, CA) or anti-FLAG M2 mAb (Sigma) and protein A-Sepharose CL-4B beads (Pharmacia). For Western blot analysis, lysates containing equal amounts of total protein were resolved by 4-20% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. The blots were then probed with mAb M2, followed by biotinylated rabbit anti-mouse IgG (Amersham Life Science Inc.) and developed using the enhanced chemiluminescence (ECL) detection system (Amersham Life Science Inc.).

Cells transfected with FLAG-tagged p38 or p38 were lysed, and recombinant protein was immunoprecipitated using mAb M2 and protein A-Sepharose CL-4B beads. Beads were washed three times with lysis buffer, once with kinase buffer (25 mM HEPES, pH 7.4, 25 mM -glycerophosphate, 25 mM MgCl₂, 25 mM dithiothreitol, 0.1 mM Na₃VO₄), and resuspended in 40 µl of kinase buffer. The beads were then incubated with ATF-2 and 1 µl of [-³²P]ATP (3000 Ci/mmol) at 30 °C for 30 min. Reaction mixtures were then resuspended in 2 × sample buffer (125 mM Tris, pH 6.8, 6% SDS, 20% glycerol) and boiled for 3 min. Phosphorylated proteins were resolved by SDS-PAGE, after which the gels were dried and exposed to radiographic film.

To determine the substrate and inhibitor specificity of p38 and p38, the kinases were first activated in vitro with ca-MKK3. Activation was performed in the presence of Dulbecco's phosphate-buffered saline, pH 7.4, 10 mM MgCl₂, 0.5 mM EGTA, 0.1 mM Na₃VO₄, 50 nM calyculin A, 0.1% -mercaptoethanol, 100 µM ATP, 75 µg/ml ca-MKK3, and approximately 20 µg/ml p38 or p38. The reaction (60 min at 28 °C) was terminated by washing the p38-bound immunoprecipitates three times with Dulbecco's phosphate-buffered saline. Substrate specificity studies were performed in kinase buffer (40-µl reactions; 30 min at 28 °C) with 2 µM cold ATP, 5 µCi of [-³²P]ATP (3000 Ci/mmol), no ca-MKK3, approximately 1 µg/ml p38 or p38, and 50 µg/ml of either full-length GST-ATF-2, GST-c-Jun, GST-MAPKAP kinase-2, GST-MAPKAP kinase-3, or PHAS-I. To evaluate the sensitivities of p38 and p38 to AMG 2372, 40-µl kinase reactions (15 min at 28 °C) were performed as described for substrate reactions, except that ATF-2-(1-109) was used as substrate in the presence of various inhibitor concentrations. Reactions were terminated by boiling in the presence of 2 × sample buffer. The reaction products were resolved by SDS-PAGE, visualized by autoradiography, and quantified using a PhosphorImager (Molecular Dynamics).

RESULTS

Molecular Cloning of p38

To identify novel MAPKs, we searched the Amgen EST data base with the p38 nucleotide sequences as the query sequences. One partial human cDNA sequence (311 base pairs) was identified that has homology to p38. Through PCR and hybridization techniques, we isolated a full-length cDNA corresponding to this EST sequence. The novel cDNA is predicted to encode a protein of 365 amino acids with a molecular mass of 42 kDa (Fig. 1A). The deduced amino acid sequence predicts a protein kinase with all 11 characteristic kinase subdomains (Fig. 1B). GenBank^TM and European Molecular Biology Laboratory Data base searches identified p38/CSBP2 (14), p38 (39), and p38/ERK6 (40-42) as the most closely related molecules. p38 displays 57, 55, and 62% amino acid identity to p38/CSBP2, p38, and p38/ERK6, respectively. p38 has a putative dual phosphorylation motif (TGY) in kinase subdomain VIII similar to that found in other p38 family members. The amino acid sequence alignment of p38 with human p38/CSBP2, p38, and p38/ERK6 is shown in Fig. 1B.

Nucleotide and deduced amino acid sequences of p38 and amino acid sequence alignment of p38 with other p38 family members.

Tissue Distribution of p38 mRNA

The expression of p38 was examined in a variety of human tissues by Northern blot analysis using a probe derived from the 5 end of p38. The p38 probe hybridized strongly to a transcript of approximately 1.8 kilobases and weakly to a transcript of 6.0 kilobases, while a probe derived from p38 hybridized to a single transcript of 4.1 kilobases (14) (data not shown). The same p38 probe was used to hybridize a human RNA master blot containing poly (A)⁺ RNAs from 50 different human tissues. The RNAs in this blot have been normalized to the mRNA levels of eight different housekeeping genes; thus the relative levels of mRNA in different tissues could be determined. Among the tissues examined, strong hybridizing signals were observed in exocrine/endocrine tissues including human salivary gland, pituitary gland, adrenal gland, and placenta (Fig. 2A). Moderate signals were observed in pancreas, trachea, thyroid gland, stomach, prostate, colon, small intestine, lymph node, kidney, and lung. Probing the master blot with p38 DNA revealed a different tissue distribution profile. Strong hybridizing signals were found in placenta, cerebellum, bone marrow, thyroid gland, peripheral leukocyte, liver, and spleen. Moderate signals were found in occipital lobe, fetal liver, pituitary gland, adrenal gland, aorta, uterus, stomach, lymph node, cerebral cortex, hippocampus, and thymus (Fig. 2B). Probing the master blot with p38 DNA found that p38 is abundantly expressed in brain tissues such as hippocampus, frontal lobe, cerebral cortex, cerebellum, caudate nucleus, medulla oblongata, whole brain, and fetal brain (Fig. 2C). Interestingly, probing the master blot with p38 DNA found that it has a very limited tissue distribution profile. p38 was highly expressed in skeletal muscle, while the expression in other tissues appears to be low (Fig. 2D).

Expression pattern of human p38 and p38 mRNA.

Substrate Specificity of p38

Full-length p38 cDNA and p38 were cloned into mammalian expression vectors with a FLAG epitope sequence added at the 5 end and transfected into 293 cells. Transfected cell lysates were subjected to immunoprecipitation with a FLAG mAb. The immunoprecipitated p38 and p38 were activated using recombinant ca-MKK3, washed, and used in immune complex kinase assays with various substrates. As shown in Fig. 3, p38 and p38 phosphorylated full-length ATF-2 (lanes 1 and 6) and PHAS-I (lanes 5 and 10), but not c-Jun (lanes 2 and 7), a known substrate for JNK (6). We also observed that p38 showed minimal phosphorylating activity against MAPKAP kinase-2 and -3 (Fig. 3, lanes 3 and 4), while p38 phosphorylated these substrates efficiently (Fig. 3, lanes 8 and 9). Control lysates did not phosphorylate any of the substrates (data not shown).

Substrate specificity of human p38.

Activation of p38 by Extracellular Stimuli

The p38 group of kinases can be activated by a variety of stress stimuli and proinflammatory cytokines (13, 14, 18). Because p38 is closely related to p38, we determined whether similar stimuli could activate p38 kinase activity. 293 cells were transiently transfected with either p38 or p38 cDNA and treated with various stimuli. p38 and p38 activity was measured by their ability to phosphorylate ATF-2-(1-109) in an immune complex assay. As shown in Fig. 4A, p38 was strongly activated by H₂O₂, UV, NaCl, and Na₃VO₄ and moderately activated by anisomycin, IL-1, TNF, and epidermal growth factor. p38 was strongly activated by UV, NaCl, H₂O₂, and anisomycin and moderately activated by TNF, IL-1, and epidermal growth factor (C). A notable difference is that Na₃VO₄ strongly activated p38 (Fig. 4A, lane 5) but not p38 (Fig. 4C, lane 5). To eliminate the possibility that changes in p38 kinase activity are due to the variations in protein expression, Western blot analysis was performed. Fig. 4, B and D, shows that similar amounts of p38 were expressed under all conditions tested.

Activation of p38 by extracellular stimuli in 293 cells.

Activation of p38 by Upstream Mitogen-activated Kinase Kinases

MAPKs are activated by upstream MKK kinases. To determine which MKK(s) can activate p38, we co-transfected 293 cells with vectors encoding p38 and MKK3, MKK4, or MKK6 and then assayed p38 kinase activity in an immune complex assay. In repeated experiments, co-transfection of cells with p38 and MKK3 or MKK6 resulted in strong activation of p38 activity (Fig. 5A, lanes 2 and 4), whereas co-transfection of p38 with MKK4 had little effect (Fig. 5A, lane 3). Similar studies showed that MKK6 and MKK4 markedly activated p38 (Fig. 5C, lanes 3 and 4), while MKK3 weakly activated p38 (Fig. 5C, lane 2). Western blot analysis indicated that p38 and p38 were expressed at similar levels under all conditions tested (Fig. 5, B and D).

Activation of p38 by upstream protein kinases.

p38 Is Activated by Phosphorylation at the Dual Phosphorylation TGY Motif

MAPKs are activated by dual phosphorylation at the Thr-Xaa-Tyr motif within kinase subdomain VIII (18). To determine whether this motif is required for p38 activation, we generated a mutant p38 by replacing the Thr-Gly-Tyr motif with Ala-Gly-Phe (AGF mutant) and tested whether this mutant could be activated. Wild type p38 phosphorylated ATF-2 when activated by UV irradiation (Fig. 6A, lane 4) or by co-transfection with MKK6 (Fig. 6A, lane 7). However, the AGF mutant was unresponsive to UV stimulation (Fig. 6A, lane 6) or activation by upstream kinase MKK6 (Fig. 6A, lane 8). Western blot analysis demonstrated that the AGF mutant was expressed to comparable levels as wild type p38 (Fig. 6B).

Effect of AGF mutation on the activation of p38.

Effect of AMG 2372 on p38 and p38 Kinase Activity in Vitro

p38 and p38 from transfected cell lysates were immunoprecipitated with mAb M2 and activated in vitro using purified ca-MKK3. Excess ca-MKK3 was used to ensure that p38 and p38 were maximally activated. Kinase assays were then performed in the presence of the indicated concentrations of AMG 2372. AMG 2372 only weakly inhibited the kinase activity of p38 (Fig. 7A), whereas p38 kinase activity was inhibited in a dose-dependent manner (Fig. 7B). At 1 µM inhibitor concentration, there was 98% inhibition of p38 (Fig. 7B, lane 3), but less than 25% inhibition of p38 (Fig. 7A, lane 3). Similar results were obtained in three separate experiments.

Effect of AMG 2372 on the kinase activity of p38 and p38 in vitro.

DISCUSSION

In this report, we describe the cloning and characterization of a novel member of the p38 group of protein kinases. p38 has significant homology at the amino acid level to p38, -, and - and contains the dual phosphorylation TGY motif that is found in this p38 group of kinases (13, 18). Mutation of the Thr and Tyr residues in the TGY motif abolished the kinase activity of p38 and blocked UV or MKK6-induced activation. Thus, like other MAPKs, p38 requires phosphorylation at the Thr and/or Tyr in the TGY motif for its activation.

The tissue distribution of p38 was examined in 50 different human tissues. The pattern of expression of p38 mRNA is distinct from that of p38, -, and -. Very high levels of expression of p38 mRNA were observed in human gland tissues, while p38 was abundantly expressed in placenta, brain (cerebellum), and lymphoid tissues. p38 is most abundantly expressed in brain tissues, while p38 appears to have a limited tissue distribution. These differences in mRNA expression suggest that p38, p38, p38, and p38 may have tissue-specific functions.

Similarities among p38, p38, p38, and p38 prompted us to investigate whether p38 can utilize the same substrates. p38 phosphorylated ATF-2 and PHAS-I as efficiently as p38, but not MAPKAP kinase-2 and -3 which are the physiological p38 substrates. This is also in contrast to the substrate profile of p38 which utilize similar substrates as p38 (39). The p38 could phosphorylate ATF-2 but was also far less effective in phophorylating MAPKAP kinase-2 and -3 (42). Thus the substrate specificity of p38 resembles that of p38.

Similar to p38, p38 is activated in 293 cells by a diverse array of cellular stresses and proinflammatory cytokines. However, the degree of activation by various stimuli is different for p38 as compared with p38. Most notable was the strong activation of p38, but not of p38 by Na₃VO₄. Because Na₃VO₄ inhibits protein tyrosine phosphatase activity, our data suggest that such phosphatases differentially regulate the basal activity of p38 and p38.

Differences in activation of p38 versus p38 were also observed at the MKK level. In cell transfection experiments, p38 is strongly activated by MKK3 and MKK6, whereas p38 is preferably activated by MKK6. These data suggest that regulators of p38 overlap. Like p38, it is likely that the dominant activator of p38 in a given cell type will reflect the unique cellular environment. For example, it has been observed that MKK6 is the dominant activator of p38 in monocytes and KB cells, while MKK3 is the dominant activator of p38 in PC-12 cells (46).

p38/CSBP has been directly linked to inflammatory cytokine production through the use of inhibitors that block its function. We tested one compound that blocks p38 activity and found that it was relatively inactive against p38. Thus, other compounds will have to be developed to determine if p38 is involved in cytokine production. The critical substrates phosphorylated by p38 leading to cytokine production have not yet been elucidated, although several candidates have been discovered. Of this group, we showed that p38 phosphorylated ATF-2 and PHAS-I, but not MAPKAP kinase-2 and -3. Additional studies are required to identify in vivo p38 substrates and to determine if these substrates are involved in cytokine production or other p38-mediated processes.